In vivo two-photon imaging methods will be used to explore the mechanisms of dendrite growth and synaptogenesis in the zebrafish retinotectal pathway. The project will focus especially on effects of visually stimulated activity on dendrite growth and synaptogenesis. The zebrafish tectum is very well suited to two- photon fluorescence imaging, and development of the retinotectal projection is known to depend on activity. Novel genetically encoded fluorescent protein (FP) markers and automated two-photon (2P) microscopy will be used to efficiently study dynamics of dendritic arbor growth and synaptogenesis at high time resolution over long time periods. The research involves four specific aims: (1) determine kinetic parameters of dendrite growth and synaptogenesis under baseline (dark, unstimulated) conditions, (2) measure effects of defined visual stimuli on kinetic parameters of dendrite growth and synaptogenesis, (3) test selective glutamate receptor antagonists for effects on growth and synaptogenesis parameters under baseline and visual stimulation conditions, and (4) measure and analyze dendritic Ca transients evoked by patterned visual stimulation. These studies will be among the first to integrate dynamic imaging of dendritic arbor growth with visualization of synaptogenesis processes. Experiments based on this new capability will test various current hypotheses relating the dynamics of dendrite growth and synaptogenesis to each other and to the activity-dependent development of functional neural circuitry. To understand normal or pathological brain development, it is obviously necessary to understand dendrite growth and synaptogenesis. It now appears that dendrite growth and synaptogenesis processes may persist in mature brains, as well, so our studies of these processes in developing zebrafish may advance understanding of the plasticity and pathologies of mature nervous systems. In addition, the developmental insights obtained may point to unexpected therapeutic opportunities for l]'eating disorders of vision, and repairing effects of neurological trauma, neurodegeneration, and drug addiction.